This invention relates to signal transduction in mammalian cells.
Human hepatocellular carcinoma (HCC) is one of the most common and least understood tumors. Although persistent hepatitis B and C virus infections are major risk factors for the development of this disease, little is known regarding cellular pathogenesis. Normal hepatocyte proliferation is regulated by several growth factors, of which insulin, epidermal growth factor (EGF), transforming growth factor xcex1 (TGFxcex1) and hepatocyte growth factor (HGF) are believed to be the most important (Moradpour et al., IN: Hepatology, 3rd ed., W. B. Saunders, Philadelphia, Pa.). Such growth factors bind to hepatocyte cell surface receptors with intrinsic tyrosine kinase activity and initiate a series of protein phosphorylation events within the cells. Tyrosyl phosphorylation (TP) of downstream molecules transmits the mitogenic signals from the cell surface to the nucleus through several signal transduction pathways.
A cDNA encoding one of the key molecules involved in the insulin mediated signal transduction cascade, human insulin receptor substrate-1 (human IRS-1 or hIRS-1), has been found to be overexpressed at the protein and RNA levels in HCC cell lines and tumor tissues (Furusaka et al., Mol. Cell Biol. 11:4405-4414, 1991; Nishiyama et al., Biochem. Biophys. Res. Commun. 183:280-285, 1992). Tyrosine residues of IRS-1 are phosphorylated following cellular stimulation by ligands such as insulin; insulin-like-growth-factor 1 (IGF-1); interleukins 4, 9 and 13; interferons xcex1 and xcex2; growth hormone; leukemia inhibitory factor; and tumor necrosis factor (Artersinger et al., J. Biol. Chem. 270:14685-14692, 1995; Guo et al., J. Biol. Chem. 271:615-618, 1996; Myers et al., J. Biol. Chem. 270:11715-11718, 1995; Platanias et al., J. Biol. Chem. 271:278-282, 1996; Welham et al., J. Biol. Chem. 270:12286-12296, 1995). Tyrosyl phosphorylated IRS-1 serves as a key xe2x80x9cdockingxe2x80x9d protein. It transmits mitogenic or metabolic signals by interacting, through specific motifs, with downstream molecules containing the Src homology domain 2 (SH2) (Sun et al., Nature 377:173-177, 1995). For example, the 897YVNI (SEQ ID NO:1) motif of hIRS-1 binds to the Grb2 adapter protein (Baltensperger et al., Science 260:1950-1952, 1993); the 1180YIDL (SEQ ID NO:2) motif binds to Syp phosphatase (also known as PTP1D, PTP2C, and SH-PTP2) (Kuhne et al., J. Biol. Chem. 268:11479-11481, 1993); and 613YMPM (SEQ ID NO:3) and 942YMKM (SEQ ID NO:4) motifs are the principal binding sites for the p85 subunit of phosphatidylinositol-3 kinase (PI3K) (Backer et al., EMBO J. 11:3469-3479, 1992; Myers et al., Proc. Natl. Acad. Sci. USA 89:10350-10354, 1992). While TP sites are recognized throughout the entire IRS-1 protein, the SH2-binding domains are located only in the C-terminal region (Myers et al., Trends Biochem. Sci. 19:289-293, 1994). The N-terminal sequences, however, contain three important functional domains identified as a pleckstrin homology (PH) region, located at amino acid residues 9-117 (Musacchio et al., Trends Biochem. Sci. 18:343-348, 1993), and two regions homologous to a phosphotyrosine binding (PTB) domain, located at amino acid residues 161-317 (Sun et al., Nature 377:173-177, 1995) and at amino acid residues 314-463 (Gustafson et al., Mol. Cell. Biol. 15:2500-2508, 1995).
Normal hepatic growth has been associated with TP of IRS-1 and its subsequent interaction with SH2-containing molecules such as Grb2 and PI3K during the G1 phase of the hepatocyte cell cycle following partial hepatectomy (Sasaki et al., J. Biol. Chem. 268:3805-3808, 1993). There is now evidence to support the hypothesis that IRS-1 may have transforming properties as well (D""Ambrosio et al., Cell. Growth Differ. 6:557-562, 1995; Ito et al., Mol. Cell. Biol. 16:943-951, 1996). Stable transfection and overexpression of the hIRS-1 gene in NIH 3T3 cells leads to increased TP of the protein, enhanced binding of the protein to Grb2 and Syp but not PI3K, and persistent activation of the downstream MAPK (mitogen-activated protein kinase) cascade. Such transfected cells develop a phenotype characterized by increased transformed foci formation, induction of anchorage independent cell growth, increased cell proliferation and formation of large tumors in nude mice (Ito et al., Mol. Cell Biol. 16:943-951, 1996). The functional domains of the hIRS-1 protein required for its transforming activity have been shown to reside in both the 897YVNI (SEQ ID NO:2) and 1180YIDL (SEQ ID NO:3) motifs (Tanaka et al., J. Biol. Chem., 1996). Insulin and IGF-1 have been shown to act as dominant cellular mitogens for several different human tumors including HCC (Macaulay, Br. J. Cancer 65:311-320, 1992).
The present invention is based on the discovery of the functional domains of mammalian IRS-1""s that are essential for insulin- and IGF-1-induced TP and for subsequent activation of downstream signal transduction molecules associated with tumorigenicity. Applicants have also discovered that a dominant negative mutant protein derived from hIRS-1 blocks TP of endogenous hIRS-1 protein and other substrates of insulin- or IGF-1- induced TP (e.g., Shc). This mutant protein also reverses the malignant phenotype of HCC cells.
Accordingly, the invention features dominant negative mutants of mammalian (e.g., human) IRS-1 proteins. The mutants, when co-expressed with a wild type IRS-1 in a cell, block the function of the wild type IRS-1 in the cell. Dominant negative mutants substitute for wild-type proteins implicated in pathogenicity and can counteract their pathogenic effects.
The mutants of the invention can contain the pleckstrin homology domain and the two phosphotyrosine binding domains of their corresponding wild type IRS-1""s. For instance, a dominant negative mutant of human IRS-1 can contain 460 amino acid residues from the amino-terminal half (i.e., amino acid residues 1-621) of native IRS-1. The pleckstrin homology (PH) domain corresponds approximately to amino acid residues 9-117 of hIRS-1, and is believed to bring IRS-1 to close proximity to cell membranes on which insulin receptor resides. The two phosphotyrosine domains (PTB) correspond approximately to amino acid residues 161-314 and 315-463 of hIRS-1, respectively. The PTB domains, when bound to insulin receptor, become phosphorylated at their tyrosine residues. IRS-1 thus phosphorylated can transmit the signal from the insulin receptor via the IRS-1xe2x80x2 SH2-binding region to proteins residing downstream in the insulin/IGF-1 signal transduction pathway.
The mutant protein may also lack at least one or even all of the functional SH2-binding motifs at its SH2-binding region, so that the protein can no longer bind to the SH2 domain of one or all of its adaptor proteins (e.g., Syp, Grb2, PI3K, or NCK). For instance, a dominant negative mutant of human IRS-1 may lack at least 300 amino acid residues from its carboxy-terminal half (i.e., amino acid residues 622-1243). It may even lack the last 727 amino acid residues of human IRS-1.
The mutant can additionally contain a heterologous sequence, i.e., a sequence not related to IRS-1, to facilitate its identification or purification. The heterologous sequence may contain an epitope to which an antibody can bind, or a ligand (e.g., a maltose-binding protein domain or a His tag) of any other receptor molecule (e.g., maltose or nickel). This sequence can range from, e.g., 4-25 amino acid residues in length, and replace 0-25 amino acid residues of the IRS-1 sequence in the mutant protein. It will typically be at one end of the mutant protein, but can be in the middle. Exemplary epitopes include FLAG, E-tag, c-myc tag, VSV-GP, T7 tag, HSV tag, and HA tag, all of which are well known in the art. The identity of the tag sequence is not critical.
Due to polymorphism that may exist at the IRS-1 genetic locus, minor variations in the amino acid sequence of the IRS-1""s found in any given mammalian species may occur. For purposes of this invention, as long as adequate dominant negative effect on wild type IRS-1 remains, mutant proteins containing minor amino acid sequence variations as a result of natural IRS-1 polymorphism, or even as a result of recombinant genetic manipulation, are within the scope of this invention. In particular, one or all of the region corresponding to residues 118-160 of hIRS-1 (i.e., between the PH domain and the closest PTB domain) can be deleted or substituted with alternative residues. Preferably 0-35 of those residues would be deleted or substituted, and more preferably 0-25 (e.g., 0-10 or 0-5).
This invention also includes methods of inhibiting tyrosyl phosphorylation of an IRS-1 in a mammalian cell (e.g., a human hepatic cell). In these methods, an effective amount of a mutant of the invention is introduced into the cell (e.g., by expression from a recombinant expression construct within the cell). An effective amount is an amount that decreases the insulin- or IGF-1-induced TP of the endogenous IRS-1.
The dominant negative mutants of the invention can be used to reverse (e.g., eliminate or mitigate) a malignant phenotype of mammalian tumors (e.g., brain cancer, lung cancer, pancreatic cancer, and gastrointestinal cancers such as colon, liver cancer, or stomach cancer) in which hyperactivity of IRS-1 is known or believed to play a causative role. The malignant phenotype can be characterized by, e.g., growth without contact inhibition, anchorage-independent growth, increased rate of proliferation as compared to a normal cell, or ability to form a tumor in a nude mouse.
Also within the scope of the invention are therapeutic compositions containing a polypeptide of the invention, or a DNA encoding the polypeptide, admixed with a pharmaceutically acceptable carrier.
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Exemplary methods and materials are described below, although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. The materials, methods, and examples are illustrative only and not intended to be limiting.
Other features and advantages of the invention will be apparent from the following detailed description, and from the claims.